(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
5 June 2003 (05.06.2003)
PCT
(10) International Publication Number
WO 03/046207 A2
(51) International Patent Classification 7 : C12Q
(21) International Application Number: PCT/US02/38434
(22) International Filing Date:
26 November 2002 (26.1 1.2002)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/333,884 27 November 2001 (27.1 1.2001) US
(71) Applicant (for all designated States except US): FRED
HUTCHINSON CANCER RESEARCH CENTER
[US/US]; Office of Technology Transfer, 1100 Fairview
Avenue North, M/S C2M 027, Seattle, WA 98109-1024
(US).
(72) Inventors; and
(75) Inventors/Applicants (for US only): BEDALOV, Anto-
nio [HR/US]; 3009 South Washington Street, Seattle, WA
98144 (US). GOTTSCHLING, Daniel, E. [US/US]; 209
NW 48lh Street, Seattle, WA 98107 (US). SIMON, Julian
[US/US]; 5300 South Hudson Street, Seattle, WA 98118
(US).
(74) Agents: KEZER, William, B. et al.; Townsend and
Townsend and Crew LLP, Two Embarcadero Center,
Eighth Floor, San Francisco, CA 94111-3834 (US).
(81) Designated States (national): AU, CA, JP, US.
(84) Designated States (regional): European patent (AT, BE,
BG, CI I, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE, SK, TR).
Declaration under Rule 4.17:
— of inventorship (Rule 4. 1 7(iv)) for US only
Published:
— without international search report and to be republished
upon receipt of that report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
= (54) Title: METHODS FOR INHIBITING DEACETYLASE ACTIVITY
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^ (57) Abstract: A method for identifying a compound that inhibits the NAD+ -dependent dcacctylase activity of a SIR2 protein is
disclosed. These compounds are useful for the treatment of cancers and other diseases, through the activation of silenced genes,
^ through the promotion of apoptosis in cancerous cells, and through the inhibition of transcriptional repressor activity in oncogenes.
WO 03/046207
PCT/US02/38434
METHODS FOR INHIBITING DEACETYLASE ACTIVITY
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States Provisional Application
5 No. 60/333,884, filed November 27, 2001 . The foregoing application is hereby incorporated
by reference in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
1 0 [0002] A portion of the present invention was made under federally sponsored research and
development under National Heart, Lung, and Blood Institute Grant HL0421 1, National
Institutes of Health Grant GM43893, and National Cancer Institute Grant CA78746. The
Government may have rights in certain aspects of this invention.
1 5 REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER
PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK.
[0003J NOT APPLICABLE
BACKGROUND OF THE INVENTION
20 [0004J Portions of the eukaryotic genome can be maintained in a transcriptionally inactive,
or silenced, state as the result of the local chromatin structure. Silent chromatin may
encompass regions ranging from a few thousand base pairs, as in the silent mating type genes
of the yeast S. cerevisiae (Loo, S. & Rine, J. (1995) Annu. Rev. Cell Dev. Biol. 11, 519-48),
to whole chromosomes, such as the inactive X-chromosome in mammals (Lyon, M. F. (1999)
25 Curr. Biol. 9, R235-7). The formation of silent chromatin, which is best understood at the S.
cerevisiae silent mating type loci HMR and HML y and telomeres, depends on DNA elements,
or silencers. The HM silencers are located in proximity to the genes they regulate and contain
a combination of binding sites for Raplp, Abflp and the origin recognition complex (ORC)
(Loo, S. & Rine, J. (1995) Annu. Rev. Cell Dev. Biol. 11, 5 19-48). These proteins recruit the
30 SIR (Silent Information Regulator) protein complex (Sir2p-4p) through protein-protein
interactions. Once recruited to silencers, the SIR complex is thought to spread along the
chromatin through binding of Sir3p and Sir4p to the NH 2 -terminal tails of histone H3 and H4
(reviewed in Gartenberg, M R. (2000) Curr. Opin. Microbiol. 3, 132-7). Among the many
1
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PCT/US02/38434
requirements for silent chromatin (reviewed in Wu, J. & Grunstein, M. (2000) Trends
Biochem. Sci. 25, 619-23), post-translational modification (i.e. acetylation, phosphorylation,
methylation and ubiquitination) of the NH2-terminal tails of histones appears to be critical.
For example, the tails of histones H3 and H4 are hypoacetylated in silent chromatin
5 compared to other regions of the genome (Braunstein, M. et al. (1993) Genes Dev. 7, 592-
604). Of the SIR proteins, Sir2p has been shown to be an NAD + -dependent histone
deacetylase, and is responsible for the hypoacetylated state of histones in silent chromatin
(Moazed, D. (2001) Curr. Opin. Cell Biol 13, 232-8; Imai, S. et al. (2000) Nature 403, 795-
800; Smith, J. S. et al (2000) Proc. Natl Acad. Sci. USA 97, 6658-63; Landry, J, et al
10 (2000) Proc. Natl Acad. Sci. USA 97, 5807-1 1). Sir2p also acts at the ribosomal RNA
gene cluster (rDNA) in the RENT protein complex, which does not include Sir3p or Sir4p
(Straight, A. F. et al (1999) Cell 97, 245-56), where it acts to repress recombination.
(0005] The yeast SIR2 gene is the defining member of a broadly conserved family of
NAD + -dependent deacetylases, termed sirtuins, found in organisms ranging from bacteria to
1 5 humans (Frye, R. A. (2000) Biochem. Biophys. Res. Commun. 273, 793-8). Sirtuins are
highly conserved and contain a conserved catalytic domain of approximately 275 amino acids
(Grozinger, CM. et al, (2001) J. Biol Chem. 276, 38837-38843). In S. cerevisiae alone,
four additional homologies have been identified, while in humans, eight homologues have
been identified (Grozinger, CM. et al (2001) supra). The yeast SER2 gene shares the
20 greatest similarity with genes found in other eukaryotes, where it is believed that these
closely related homologues serve a comparable role in silencing. Interestingly, SIR2 and its
homologues have been implicated in the genetic regulation of aging, both in yeast and C.
elegans (Tissenbaum, H. A. & Guarente, L. (2001) Nature 410, 227-30; Sinclair, D. A. &
Guarente, L. (1997) Cell 91, 1033-42) and in metazoan development though the details of
25 how it affects these fundamental processes are still mysterious.
[0006] Recently, several groups (Luo, J. et al (2001) Cell 107, 137-48; and Vaziri, H. et al
(2001) Cell 107, 149-59) have explored the influence of the mammalian homologues, Sir2a
(the mouse homologue of S. cerevisiae SIR2, also known as mSIRTl) and SIR2a (the human
homologue of S. cerevisiae SIR2, also known as hSIRTl\ on the activity of the p53 tumor
30 suppressor gene. These studies indicate that deacetylase activity of Sir2a and SIR2a act on
p53, resulting in suppression of the tumor suppressor activity. They have also shown that this
deacetylase activity is dependent on nicotinamide adenosine dinucleotide (NAD).
2
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[0007] What is needed in the art, is a method for inhibiting the NAD + -dependent
deacetylase activity of a member of the SIR2 family of proteins using a small molecule.
Surprisingly, the present invention meets this need.
5 BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides a method for identifying compounds
useful for the treatment of cancer or genetic blood diseases, comprising the step of
determining whether the compound inhibits the deacetylase activity of a NAD + -dependent
deacetylase. In a related aspect of the present invention, the method for treating cancer or
10 genetic blood diseases comprises the step of administering to a subject in need thereof, a
therapeutically effective amount of a compound that inhibits the deacetylase activity of a
NAD + -dependent deacetylase.
[0009] In a second aspect of the present invention, a method is provided for identifying
compounds which will be useful for the treatment of cancer or genetic blood diseases,
1 5 comprising the step of determining whether the compound inhibits the NAD + -dependent
deacetylase activity of a member of the SIR2 family of proteins. In a related aspect of the
present invention, the method for treating cancer or genetic blood diseases comprises the step
of administering to a subject in thereof, a therapeutically effective amount of a compound
that inhibits the NAD + -dependent deacetylase activity of a member of the SIR2 family of
20 proteins.
[0010] In a third aspect of the present invention, a method is provided for activating a
silenced gene in a cell, comprising contacting the cell with an effective amount of a
compound which is capable of inhibiting the NAD + -dependent deacetylase activity of a
member of the SIR2 family of proteins.
25 [001 1] In a fourth aspect of the present invention, a method is provided for promoting p53-
dependent apoptosis of a cell comprising contacting the cell with an effective amount of a
compound which is capable of inhibiting the NAD + -dependent deacetylase activity of a
member of the SIR2 family of proteins.
[0012] In a further aspect of the present invention, a method is provided for inhibiting
30 BCL6 transcriptional repressor activity, comprising contacting a cell with an effective
amount of a compound which is capable of inhibiting the NAD + -dependent deacetylase
activity of a member of the SIR2 family of proteins.
[0013] In another aspect of the present invention, a method is provided for inhibiting the
deacetylase activity of a NAD + -dependent deacetylase comprising contacting the
3
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NAD + -dependent deacetylase with a NAD + -dependent deacetylase inhibiting amount of a
compound of Formula I:
In Formula I, the letter X is a member selected from the group consisting of 0 and S. The
5 symbols L 1 and L 2 each represent members independently selected from the group consisting
symbols L l and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of Ct^alkyl, C 2 -6alkenyl and -C0 2 R 4 . The symbols R* and R 2 each
1 0 represent members independently selected from the group consisting of hydrogen,
C^alkoxy, Co^alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
The symbol R 3 represents a member selected from the group consisting of hydrogen,
C|. 6 alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
1 5 instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Ci^alkyl.
[0014] In a further aspect of the present invention, a method is provided for inhibiting the
deacetylase activity of a NAD + -dependent deacetylase comprising contacting the
NAD + -dependent deacetylase with a NAD + -dependent deacetylase inhibiting amount of a
20 compound of Formula II :
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR e , -NR C R C , -C0 2 R c , -C(0)R e , -C(0)NR c R c , -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
X
I
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
25
4
WO 03/046207
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In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR e , while the symbol R° represents a member selected from the
group consisting of hydrogen, Ci^alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci. 6 alkyl, aryl, -OR e , -NR e R e , -CN, -N0 2 and
5 halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Cu 6 alkyl, aryl, -OR c , -NR e R e and halogen. And, each instance of the symbol R e
independently represents a member selected from the group consisting of hydrogen and
C^alkyl.
[0015] In a further aspect of the present invention, a method is provided for the treatment
10 . of cancer comprising the step of administering to a subject in need of such treatment a first
amount of an antineoplastic agent and a second amount of a compound of Formula I:
I
In Formula I, the letter X is a member selected from the group consisting of O and S. The
symbols L 1 and L 2 each represent members independently selected from the group consisting
15 of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of Ci^alkyl, C2^alkenyl and -CO2R 4 . The symbols R 1 and R 2 each
represent members independently selected from the group consisting of hydrogen,
20 Ci^alkoxy, Co-ealkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
The symbol R 3 represents a member selected from the group consisting of hydrogen,
C,. 6 alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
instance of the symbol R 4 independently represents a member selected from the group
25 consisting of hydrogen and Ci^alkyl.
[001 6] In another aspect of the present invention, a method is provided for the treatment of
cancer comprising the step of administering to a subject in need of such treatment a first
amount of a an antineoplastic agent, and a second amount of a compound of Formula II:
5
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II
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C,_ 6 alkyl, aryl, -OR e , -NR c R e , -C0 2 R c , -C(0)R c , -QOJNR'R 6 , -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ci-6alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR e , -NR e R e , -CN, -N0 2 and
10 halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Ci^alkyl, aryl, -OR c , -NR e R c and halogen. And, each instance of the symbol R e
independently represents a member selected from the group consisting of hydrogen and
Ci^alkyl.
[001 7J In yet another aspect of the present invention, a composition is provided for the
15 treatment of cancer comprising an antineoplastic agent and a compound of Formula I:
In Formula I, the letter X is a member selected from the group consisting of O and S. The
symbols L 1 and L 2 each represent members independently selected from the group consisting
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
20 symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of d. 6 alkyl, C 2 ^alkenyl and -C0 2 R 4 . The symbols R 1 and R 2 each
represent members independently selected from the group consisting of hydrogen,
Ci^alkoxy, C 0 -6alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
25 together with the carbons to which they are attached to form a six-membered lactone ring.
O
6
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The symbol R represents a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Ci. 6 alkyl.
5 [0018] In a further aspect of the present invention, a composition is provided for the
treatment of cancer comprising an antineoplastic agent and a compound of Formula II:
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR e , -NR'R 6 , -C0 2 R c , -C(0)R e , -C(0)NR e R c , -CN, -N0 2 and halogen, while
10 the symbol R b is a member selected from the group consisting of:
In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ci^alkyl and aryl optionally substituted with a member
15 selected from the group consisting of hydrogen, C^alkyl, aryl, -OR c , -NR c R e , -CN, -N0 2 and
halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Ci^alkyl, aryl, -OR e , -NR e R e and halogen. And, each instance of the symbol R e
independently represents a member selected from the group consisting of hydrogen and
C^alkyl.
20 [0019] In another aspect of the present invention, a pharmaceutical composition is
provided, comprising a pharmaceutically acceptable excipient and a compound, and all
pharmaceutically acceptable salts thereof, of Formula I:
In Formula I, the letter X is a member selected from the group consisting of O and S. The
25 symbols L 1 and L 2 each represent members independently selected from the group consisting
7
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of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one~of the
symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of C^alkyl, C 2 _ 6 alkenyl and -C0 2 R 4 . The symbols R l and R 2 each
5 represent members independently selected from the group consisting of hydrogen,
Ci. 6 alkoxy, C 0 -6alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
The symbol R 3 represents a member selected from the group consisting of hydrogen,
C,. 6 alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
10 instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and C i ^alkyl.
[0020] In another aspect of the present invention, a pharmaceutical composition is
provided, comprising a pharmaceutically acceptable excipient and a compound, and all
pharmaceutically acceptable salts thereof, of Formula II:
15
II
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR c , -NR^, -C0 2 R c , -C(0)R c , -CCONR'R 6 , -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
& and vv xa
R d
O
20 In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ci-aalkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, C^alkyl, aryl, -OR e , -NR e R e , -CN, -N0 2 and
halogen. The symbol R d represents a member selected from the group consisting of
25 hydrogen, Ci. 6 alkyl, aryl, -OR e , -NRT* 6 and halogen. And, each instance of the symbol R c
independently represents a member selected from the group consisting of hydrogen and
Ci^alkyl.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Fig. 1. (A) Chemical structure of SI . (B) Activation of a TRP1 reporter at the silent
HMR mating locus by SI (S). Wild type (SIR2) or sir2A cells with TRP1 integrated into
HMR, Cells were replica plated onto complete synthetic media, or media lacking tryptophan
5 (-trp) without or with the indicated concentrations of S 1 . (C) Loss of responsiveness to a
factor in the presence of S 1 . The halo of cells indicates those able to grow. (D) S 1 increases
recombination of an ADE2 reporter integrated within ribosomal DNA array.
[0022] Fig. 2. SI -treated wild type (wt) cells and sir2A cells display similar transcriptional
changes relative to untreated wt cells. (A) Correlation of transcriptional changes between
10 genetic and chemical inactivation of Sir2p. The correlation plot shows transcriptional
changes in a sir2A mutant relative to wt (sir2A/wi) on the vertical axis and changes in wt
cells treated with SI relative to untreated wt cells (15 \xM Sl/no treatment) on the horizontal
axis. (B) A Venn diagram comparing genes up-regulated (LEFT) and down-regulated
(RIGHT) more than 2-fold relative to wt or untreated cells and sir2A 9 hstlA or SI -treated wt
1 5 cells. (Q Correlation of transcriptional changes in wt cells in response to S 1 treatment with
and without cycloheximide. The correlation plot shows transcriptional changes in a SI and
cycloheximide-treated wt cells relative to cells treated with cycloheximide alone (15 nM SI
CYH/CYH) on the vertical axis and changes in wt cells treated with SI relative to untreated
wt cells (15 jiM Sl/no treatment) on the horizontal axis. (D) Venn diagrams comparing
20 transcriptional changes (up- or down-regulation) in hst2A, hst3A hst4A cells and SI -treated
cells (split).
[0023] Fig, 3. (A) Inhibition of NAD-dependent histone deacetylase activity (HDA) of
Sir2p by S 1 . (B) Immunoblot of Sir2p in whole cell lysates containing overexpressed wild
type or drug resistant mutant SIR2. (C) Telomeric silencing in SIR2, sir2A and drug-resistant
25 SIR2 mutants. (D) Sequence alignment between yeast Sir2p and Hstl-4p. The region
displayed in the alignment contains the putative substrate-binding site. Arrows indicate the
positions of residues that, when mutated in Sir2p, confer SI resistance.
[0024] Fig. 4. (A) Cell cycle analysis of a factor arrested MATu cells treated with SI . (B)
<x2 mRNA expression from the silent HML locus in Gl -arrested cells treated with SI . The
30 RNA from MATa and MAT* sir2A cells is included for comparison. The weak lower
molecular weight band is due to cross hybridization to a2 mRNA.
[0025] Fig, 5. SI sensitizes mammalian cells to DNA damaging agents. The bar graph
inset shows viability of cells treated with SI relative to vehicle treated control.
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[0026] Fig. 6. Table of exemplary compounds of the present invention, and their potency
for the inhibition of the NAD + -dependent deacetylase activity of a member of the SIR2
family of proteins.
5 DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0027] Unless defined otherwise, all technical and scientific terms used herein generally
have the same meaning as commonly understood by one of ordinary skill in the art to which
this invention belongs. Generally, the nomenclature used herein and the laboratory
10 procedures for organic and analytical chemistry are those well known and commonly
employed in the art.
[0028] The term "alkyl ," by itself or as part of another substituent, means, unless otherwise
stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof,
which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent
15 radicals, having the number of carbon atoms designated (i.e. Ci-Cio means one to ten
carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-
octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or
20 triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-
isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl,
3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted,
is also meant to include those derivatives of alkyl defined in more detail below as
"heteroalkyl." Preferred alkyl groups are limited to hydrocarbon groups, and may be
25 branched- or straight-chain. More preferred alkyl groups are unsubstituted.
[0029] The term "alkylene" by itself or as part of another substituent means a divalent
radical derived from an alkane, as exemplified by -CH2CH2CH2CH2-, and further includes
those groups described below as "heteroalkylene." Typically, an alkyl (or alkylene) group
will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms
30 being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
[0030] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their
conventional sense, and refer to those alkyl groups attached to the remainder of the molecule
via an oxygen atom, an amino group, or a sulfur atom, respectively.
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(0031 J The term "heteroalkyl," by itself or in combination with another term, means, unless
otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or
combinations thereof, consisting of the stated number of carbon atoms and from one to three
heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen
5 and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be
quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the
heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group,
including the position at which the alkyl group is attached to the remainder of the molecule.
Examples include -CH 2 -CH 2 -0-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S-
10 CH 2 -CH 3 , -CH 2 -CH 2 ,-S(0)-CH 3 , -CH 2 -CH 2 -S(0) 2 -CH 3 , -CH=CH-0-CH 3 , -Si(CH 3 ) 3 , -CH 2 -
CH=N-OCH 3 , and -CH=CH-N(CH 3 )-CH 3 . Up to two heteroatoms may be consecutive, such
as, for example, -CH 2 -NH-OCH 3 and -CH 2 -0-Si(CH 3 ) 3 . Similarly, the term "heteroalkylene"
by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as
exemplified by -CH 2 -CH 2 -S-CH 2 CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -. For heteroalkylene
15 groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyl eneoxy,
alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and
heteroalkylene linking groups, no orientation of the linking group is implied.
[0032] The terms "halo" or "halogen," by themselves or as part of another substituent,
mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally,
20 terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For
example, the term "halo(C r C 4 )alkyr is mean to include trifluoromethyl, 2,2,2-trifluoroethyl,
4-chlorobutyl, 3-bromopropyl, and the like.
|0033] The term "aryl" means, unless otherwise stated, a polyunsaturated, typically
aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three
25 rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to four heteroatoms selected from N, O, and S,
wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule
through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl,
30 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-
imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,
2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-
benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-
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quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above
noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents
described below.
[0034J For brevity, the term "aryl" when used in combination with other terms (e.g.,
5 aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is
attached to an alkyl group {e.g., benzyl, phenethyl, pyridylmethyl and the like) including
those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-
10 naphthyloxy)propyl, and the like).
[0035] Each of the above terms (e.g. , "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are
meant to include both substituted and unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0036] Substituents for the alkyl and heteroalkyl radicals (including those groups often
15 referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected
from: -OR', =0, =NR\ =N-OR\ -NR'R", -SR\ -halogen, -SiR'R"R"', -OC(0)R\ -C(0)R\ -
C0 2 R\ -CONR'R", -OC(0)NR'R", -NR"C(0)R\ -NR'-C(0)NR"R"\ -NR"C(0) 2 R', -NH-
C(NH 2 )=NH, -NR'C(NH 2 )=NH, -NH-C(NH 2 >=NR\ -S(0)R\ -S(0) 2 R\ -S(0) 2 NR'R", -CN
20 and -N0 2 in a number ranging from zero to (2m'+l), where m* is the total number of carbon
atoms in such radical. R\ R" and R"' each independently refer to hydrogen, unsubstituted
(Ci-C 8 )alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,
unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(d-C 4 )alkyl groups. When R' and
R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to
25 form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl
and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will
understand that the term "alkyl" is meant to include groups such as haloalkyl (e.g., -CF 3 and -
CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 0CH 3 , and the like). Preferably,
substituted alkyl groups are those having 3, 2 or 1 substituents selected from the group
30 consisting of -OR', -NR'R", -halogen, -C(0)R\ -C0 2 R\ -CONR'R", -CN and -N0 2 .
[0037] Similarly, substituents for the aryl and heteroaryl groups are varied and are selected
from: -halogen, -OR', -OC(0)R\ -NR'R", -SR', -R\ -CN, -N0 2 , -C0 2 R\ -CONR'R",
-C(0)R\ -OC(0)NR'R", -NR"C(0)R\ -NR"C(0) 2 R\ ,-NR'-C(0)NR"R"\
-NH-C(NH 2 )=NH, -NR'C(NH 2 )=NH, -NH-C(NH 2 )=NR\ -S(0)R', -S(0) 2 R\ -S(0) 2 NR'R'\
12
WO 03/046207 PCT/US02/38434
-N 3 , -CH(Ph) 2 , perfluoro(CrC 4 )alkoxy, and perfluoro(C r C 4 )alkyl, in a number ranging from
zero to the total number of open valences on the aromatic ring system; and where R\ R" and
R m are independently selected from hydrogen, (C r C 8 )alkyl and heteroalkyl, unsubstituted
aryl and heteroaryl, (unsubstituted aryl)-(Ci-C 4 )alkyl, and (unsubstituted aryl)oxy-(C r
5 C 4 )alkyl. Preferably, substituted aryl groups are those having 1, 2 or 3 substituents selected
from the group consisting of -halogen, -OR\ -NR'R", -CN, -N0 2 , -C0 2 R\ -CONR'R",
-C(0)R\ -N 3 , perfluoro(C r C 4 )alkoxy, and perfluoro(C r C 4 )alkyl.
[0038] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula -T-C(0)-(CH 2 ) q -U-, wherein T and U
10 are independently -NH-, -0-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are
independently -CH r , -O-, -NH-, -S-, -S(O)-, -S(0) 2 -, -S(0) 2 NR'- or a single bond, and r is an
integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be
15 replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the
aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH 2 ) S -
X-(CH 2 ) r , where s and t are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, -
S(O)-, -S(0) 2 -, or -S(0) 2 NR'-. The substituent R' in -NR'- and -S(0) 2 NR'- is selected from
hydrogen or unsubstituted (Ci-C6)alkyl.
20 [0039] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N)
and sulfur (S).
[0040] As used herein, the term "lactone ring" refers to a five-, six- or seven-membered
cyclic ester, such as
25 [0041] The term "pharmaceutical^ acceptable salts" is meant to include salts of the active
compounds which are prepared with relatively nontoxic acids or bases, depending on the
particular substituents found on the compounds described herein. When compounds of the
present invention contain relatively acidic functionalities, base addition salts can be obtained
by contacting the neutral form of such compounds with a sufficient amount of the desired
30 base, either neat or in a suitable inert solvent. Examples of pharmaceutical^ acceptable base
addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium
13
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salt, or a similar salt. When compounds of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting the neutral form of such
compounds with a sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of pharmaceutically acceptable acid addition salts include those derived
5 from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric,
sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the
salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic,
malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-
10 tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino
acids such, as arginate and the like, and salts of organic acids like glucuronic or galactunoric
acids and the like (see, for example, Berge, S.M., et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present
invention contain both basic and acidic functionalities that allow the compounds to be
15 converted into either base or acid addition salts.
[0042] The neutral forms of the compounds may be regenerated by contacting the salt with
a base or acid and isolating the parent compound in the conventional manner. The parent
form of the compound differs from the various salt forms in certain physical properties, such
as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the
20 compound for the purposes of the present invention.
[0043] In addition to salt forms, the present invention provides compounds which are in a
prodrug form. Prodrugs of the compounds described herein are those compounds that readily
undergo chemical changes under physiological conditions to provide the compounds of the
present invention. Additionally, prodrugs can be converted to the compounds of the present
25 invention by chemical or biochemical methods in an ex vivo environment. For example,
prodrugs can be slowly converted to the compounds of the present invention when placed in a
transdermal patch reservoir with a suitable enzyme or chemical reagent.
[0044] Certain compounds of the present invention can exist in unsolvated forms as well as
solvated forms, including hydrated forms. In general, the solvated forms are equivalent to
30 unsolvated forms and are intended to be encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in multiple crystalline or
amorphous forms. In general, all physical forms are equivalent for the uses contemplated by
the present invention and are intended to be within the scope of the present invention.
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[0045] Certain compounds of the present invention possess asymmetric carbon atoms
(optical centers) or double bonds; the racemates, diastereomers, geometric isomers and
individual isomers are all intended to be encompassed within the scope of the present
invention.
5 [0046] The compounds of the present invention may also contain unnatural proportions of
atomic isotopes at one or more of the atoms that constitute such compounds. For example,
the compounds may be radiolabeled with radioactive isotopes, such as for example tritium
( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the
present invention, whether radioactive or not, are intended to be encompassed within the
1 0 scope of the present invention.
[0047] The term "SIR2" refers to the silent information regulator family of proteins, also
known as sirtuins. This family includes both mammalian and non-mammalian proteins. For
example, yeast homologies of SIR2 include, but are not limited to, HST1, HST2, HST3 and
HST4. The mammalian homologues include, but are not limited to, SIRT1, SIRT2, SIRT3,
15 SIRT4, SERT5, SIRT6, SIRT7 and SIRT8, as well as sirtuins 1 to 8. More specific examples
include, but are not limited to, Sir2p and SIR2a:
[0048] The term "NAD + -dependent deacetylase" refers to a protein that removes the acetyl
groups from a lysine residue of another protein, wherein the deacetylation is coupled to NAD
(nicotinamide adenosine dinucleotide) cleavage.
20 [0049] The term "p53-dependent apoptosis" refers to the genetically determined death of a
cell that is dependent on, or stimulated by, the p53 gene, a gene that typically inhibits non-
natural cell growth, such as that observed in tumors.
[0050] The term "BCL6 transcriptional repressor activity" refers to the activity of the
BCL6 gene that results in the repression of transcription, the process of constructing an RNA
25 chain from a DNA template.
[0051] The terms "silence", "silencing" and "silenced" refers to a mechanism by which
gene expression in particular regions of the genome are repressed.
[0052] The term "chromatin" refers to a complex mixture of nucleic acid and proteins (such
as histone) in eukaryotic cells, and is usually dispersed in the interphase nucleus and
30 condensed into chromosomes.
[0053] The term "gene" refers to any segment of DNA associated with a biological
function. Thus, genes include coding sequences and/or the regulatory sequences required for
their expression. Genes also include nonexpressed DNA segments that, for example, form
recognition sequences for other proteins.
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[0054] The term "protein" refers to series of amino acid residues connected one to the other
by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In
general, the term "protein" is used to designate a series of greater than 50 amino acid residues
connected one to the other.
5 [0055] The term "antineoplastic agent" refers to a means for inhibiting or combating the
undesirable growth of biological tissue. Antineoplastic agents include, but are not limited to,
antiangiogenic and antivascular agents, antimetabolites, antifolates and other inhibitors of
DNA synthesis, antisense oligonucleotides, biological response modifiers, DNA-alkylating
agents, DNA intercalators, DNA repair agents, growth factor receptor kinase inhibitors,
10 hormone agents, immunoconjugates, microtubule disruptors and topoisomerase I/II
inhibitors. Antineoplastic agents can also include cyclophosphamide, ;
triethylenephosphoramide, triethylenethiophosphoramide, flutamide, altretamine,
triethylenemelamine, trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-
asparaginase, BCNU, benzodepa, bleomycin, busulfan, camptothecin, capecitabine,
15 carboquone, chlorambucil, cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol,
doxorubicin, epirubicin, estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine,
hydroxyurea, ifosfamide, improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane,
mitoxantrone, novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine,
tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
20 vincristine, vinorelbine and vindesine.
[0056] The term "cancer" refers to the uncontrolled growth of abnormal cells. Specific
cancers are selected from, but not limited to, rhabdomyosarcomas, chorio carcinomas,
glioblastoma multiform as (brain tumors), bowel and gastric carcinomas, leukemias, ovarian
cancers, prostate cancers, lymphomas, osteosarcomas or cancers which have metastasized.
25 [0057] The term "genetic blood disease" refers to a hereditary disease of the blood that
includes, but is not limited to, hyperproliferative diseases, thalassaemias and sickle cell
disease.
[0058] The term "tumor suppressor gene" refers to a gene that acts to suppress the
uncontrolled growth of a cancer, such as a tumor.
30 [0059] The term "ligand binding domain" refers to a region of a protein, enzyme, or gene
that binds to a ligand selective for that particular site;
[0060] The terms "treating" and "treatment" refer to a method of alleviating or abating a
disease and/or its attendant symptoms.
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[0061 J The terms "inhibition", "inhibits" and "inhibitor" refer to a method of prohibiting a
specific action or function.
[0062] The term "therapeutically effective amount" refers to that amount of the compound
being administered sufficient to prevent development of or alleviate to some extent one or
5 more of the symptoms of the condition or disorder being treated.
[0063] The term "composition" as used herein is intended to encompass a product
comprising the specified ingredients in the specified amounts, as well as any product which
results, directly or indirectly, from combination of the specified ingredients in the specified
amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must
10 be compatible with the other ingredients of the formulation and deleterious to the recipient
thereof
[0064] The term "subject" refers to animals such as mammals, including, but not limited to,
primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the
like. In certain embodiments, the subject is a human.
15
GENERAL
[0065] The present invention involves a phenotypic screen for small molecule inhibitors of
the NAD + -dependent deacetylase activity of the SIR2 class of proteins. Several of the
proteins in this class play an important role in the silencing of genes. In one aspect, the
20 deacetylation of histone by a protein in the SIR2 class, can lead to the silencing of tumor
suppressor genes. In another aspect, the deacetylation of the p53 tumor suppressor gene by a
protein in the SER2 class, reduces p53-dependent apoptosis. Diseases in which apoptosis is
involved include diseases that are associated with an increase in cell survival due to inhibition
of apoptosis, such as cancer, autoimmune diseases, inflammatory diseases and viral infections
25 and diseases that are associated with a decrease in cell death due to hyperactive apoptosis,
such as AIDS, neurodegenerative disease, hematologic diseases, and tissue damage. A
further aspect of the present invention relates to the acetylation of BCL6 by inhibiting the
deacetylase activity of a protein in the SIR2 class. Doing so prevents expression of
differentiation genes in B-cell non-Hodgkin lymphoma (B-NHL) and diffused large B-cell
30 lymphomas (DLBCL). Therefore, inhibiting the NAD + -dependent deacetylase activity of a
protein in the SIR2 family of proteins leads to the activation of p53 and either growth or
arrest of apoptosis, it is possible to treat various cancers and disease states that are well-
known to one of skill in the art.
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EMBODIMENTS OF THE INVENTION
Methods
[0066J In view of the surprising discovery above, the present invention provides in one
aspect a method is provided for identifying compounds useful for the treatment of cancer or
5 genetic blood diseases, comprising the step of determining whether the compound inhibits the
deacetylase activity of a NAD + -dependent deacetylase. In a related aspect of the present
invention, the method for treating cancer or genetic blood diseases comprises the step of
administering to a subject in need thereof, a therapeutically effective amount of a compound
that inhibits the deacetylase activity of a NAD + -dependent deacetylase.
10 [0067] In a preferred aspect of the present invention, the identified compounds are useful
for the treatment of silenced tumor suppressor genes, B-cell-derived non-Hodgkin
lymphomas and diffuse large B-cell lymphomas. In another preferred aspect of the present
invention, the identified compounds are useful for the treatment of thalassaemias and sickle
cell disease.
1 5 [0068J In a further preferred aspect of the present invention, the step of determining
comprises the step of specifically binding radiolabeled
(l,2-dihydro-3H-naphtho[2,l-b]pyran-3-one) to the ligand binding domain of a member of
the SIR2 family of proteins.
[0069] In another preferred aspect of the present invention, the NAD*-dependent
20 deacetylase is a member of the SIR2 family of proteins. In a more preferred aspect, the
member of the SIR2 family of proteins is selected from the group consisting of Sir2p and
SIR2a. In a most preferred aspect, the member of the SIR2 family of proteins is SIR2oc
[0070] In another aspect of the present invention, a method is provided for identifying
compounds which will be useful for the treatment of cancer or genetic blood diseases,
25 comprising the step of determining whether the compound inhibits the NAD + -dependent
deacetylase activity of a member of the SIR2 family of proteins. In a preferred aspect of the
present invention, the method for treating cancer or genetic blood diseases comprises the step
of administering to a subject in thereof, a therapeutically effective amount of a compound
that inhibits the NAD + -dependent deacetylase activity of a member of the SIR2 family of
30 proteins.
[0071] In another preferred aspect of the present invention, a method is provided for
activating a silenced gene in a cell, comprising contacting the cell with an effective amount of
a compound which is capable of inhibiting the NAD + -dependent deacetylase activity of a
member of the SIR2 family of proteins.
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[0072] In still another preferred aspect of the present invention, a method is provided for
promoting p53-dependent apoptosis of a cell comprising contacting the cell with an effective
amount of a compound which is capable of inhibiting the NAD + -dependent deacetylase
activity of a member of the SIR2 family of proteins.
5 [0073] In a further aspect of the present invention, a method is provided for inhibiting
BCL6 transcriptional repressor activity, comprising contacting a cell with an effective
amount of a compound which is capable of inhibiting the NAD + -dependent deacetylase
activity of a member of the SIR2 family of proteins.
[0074] In another aspect of the present invention, a method is provided for inhibiting the
1 0 deacetylase activity of a NAD + -dependent deacetylase comprising contacting the
NAD + -dependent deacetylase with a NAD + -dependent deacetylase inhibiting amount of a
compound of Formula I:
I
In Formula I, the letter X is a member selected from the group consisting of O and S. The
15 symbols L 1 and L 2 each represent members independently selected from the group consisting
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of C^alkyl, C^alkenyl and -C0 2 R 4 . The symbols R 1 and R 2 each
20 represent members independently selected from the group consisting of hydrogen,
Ci-ealkoxy, Co^alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
The symbol R 3 represents a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
25 instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Ci^alkyl.
[0075] In a preferred aspect of the present invention, the compound of Formula I has the
following structure:
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WO 03/046207 PCT/US02/38434
R 2
In this case, the symbol R 1 is a member selected from the group consisting of hydrogen,
Ci^alkoxy and Co-6alkoxy-aryl; the symbol R 2 is a member selected from the group
consisting of hydrogen and hydroxy; the symbol R 3 is a member selected from the group
5 consisting of hydrogen and -OR 4 ; and the symbol R 4 is Ci^alkyl.
[0076] In another preferred aspect of the present invention, the symbol R 1 is a member
selected from the group consisting of Ci^alkoxy, Co^alkoxy-aryl and hydroxy. In a more
preferred aspect of the present invention, the symbol R 1 is a member selected from the group
consisting of hydroxy, methoxy and benzyloxy. In a most preferred aspect of the present
10 invention, the symbol R l is benzyloxy. In another preferred embodiment, the term aryl is a
member selected from the group consisting of phenyl and naphthyl.
[0077] In another aspect of the present invention, a method is provided for inhibiting the
deacetylase activity of a NAD + -dependent deacetylase comprising contacting the
NAD + -dependent deacetylase with a NAD + -dependent deacetylase inhibiting amount of a
1 5 compound of Formula II:
.R b
.OH
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
Ci. 6 alkyl, aryl, -OR c , -NRTl', -C0 2 R e , -C(0)R c , -CCOJNR'R', -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
and
R d
20 O
In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R° represents a member selected from the
group consisting of hydrogen, C|-6alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR c , -NR e R c , -CN, -NO2 and
25 halogen. The symbol R d represents a member selected from the group consisting of
20
WO 03/046207
PCT/US02/38434
hydrogen, Ci^alkyl, aryl, -OR c , -NR C R C and halogen. And, each instance of the symbol R c
independently represents a member selected from the group consisting of hydrogen and
C^alkyl.
[0078] In a preferred aspect of the present invention, Formula II has the following
structure:
[0079] In a further aspect of the present invention, a method is provided for the treatment
of cancer comprising administering to a subject in need of such treatment a first amount of an
antineoplastic agent and a second amount of a compound of Formula I:
In Formula I, the letter X is a member selected from the group consisting of O and S. The
symbols L l and L 2 each represent members independently selected from the group consisting
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of Ci. 6 alkyl, C^alkenyl and -C0 2 R 4 . The symbols R l and R 2 each
represent members independently selected from the group consisting of hydrogen,
C^alkoxy, Co-6alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
The symbol R 3 represents a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Ci^alkyl.
21
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[0080] In another aspect of the present invention, a method is provided for the treatment of
cancer comprising administering to a subject in need of such treatment a first amount of an
antineoplastic agent, and a second amount of a compound of Formula II:
5 In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR e , -NR € R e , -C0 2 R c , -C(0)R c , -C(0)NR e R e , -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
In the components above, the symbol X a represents a member selected from the group
10 consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ci^alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR e , -NR e R c , -CN, -N0 2 and
halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Ci^alkyl, aryl, -OR c , -NRH 6 and halogen. And, each instance of the symbol R e
15 independently represents a member selected from the group consisting of hydrogen and
Ci^alkyl.
Compositions
[0081] In yet another aspect of the present invention, a composition is provided for the
treatment of cancer comprising an antineoplastic agent and a compound of Formula I:
In Formula I, the letter X is a member selected from the group consisting of O and S. The
symbols L 1 and L 2 each represent members independently selected from the group consisting
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
25 instance of the letter R of symbols L 1 and L 2 independently represents a member selected
II
o
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PCT/US02/38434
from the group consisting of Ci^alkyl, C 2 ^alkenyl and -C0 2 R 4 . The symbols R l and R 2 each
represent members independently selected from the group consisting of hydrogen,
d^alkoxy, Co^alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken
together with the carbons to which they are attached to form a six-membered lactone ring.
5 The symbol R 3 represents a member selected from the group consisting of hydrogen,
C^alkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Cj^alkyl.
[0082] In a further aspect of the present invention, a composition is provided for the
10 treatment of cancer comprising an antineoplastic agent and a compound of Formula II:
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
C^aikyl, aryl, -OR c , -NR C R C , -C0 2 R c , -C(0)R e , -C^NR^ 6 , -CN, -N0 2 and halogen, while
In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ct^alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR c , -NR C R C , -CN, -N0 2 and
20 halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Ct^alkyl, aryl, -OR e , -NR^ 6 and halogen. And, each instance of the symbol R c
independently represents a member selected from the group consisting of hydrogen and
Ci^alkyl
[0083] In a preferred aspect of the present invention, the antineoplastic agent is a member
25 selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites,
antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological
response modifiers, DNA-alkylating agents, DNA intercalators, DNA repair agents, growth
II
the symbol R b is a member selected from the group consisting of:
15
O
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factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disruptors
and topoisomerase I/II inhibitors.
[0084] In another preferred aspect of the present invention, the antineoplastic agent is a
member selected from the group consisting of cyclophosphamide, triethylenephosphoramide,
5 triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine,
trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU,
benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil,
cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin,
estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide,
10 improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,
novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen,
temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
vincristine, vinorelbine and vindesine.
[0085] In a further preferred aspect of the present invention, the antineoplastic agent is
15 administered after the compound. In another preferred aspect, the antineoplastic agent is
administered simultaneously with the compound In yet another preferred aspect, the
antineoplastic agent is administered prior to the compound.
[0086] In another aspect of the present invention, a pharmaceutical composition is
provided, comprising a pharmaceutical^ acceptable excipient and a compound, and all
20 pharmaceutical^ acceptable salts thereof, of Formula I:
In Formula I, the letter X is a member selected from the group consisting of O and S. The
symbols L ! and L 2 each represent members independently selected from the group consisting
of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the
25 symbols L 1 and L 2 represents a member selected from the group consisting of O and S. Each
instance of the letter R of symbols L 1 and L 2 independently represents a member selected
from the group consisting of Ci^alkyl, C 2 -6alkenyl and -C0 2 R 4 . The symbols R 1 and R 2 each
represent members independently selected from the group consisting of hydrogen,
Ci-^alkoxy, Co-ealkoxy-aryl and hydroxy. Alternatively, the symbols R l and R 2 are taken
30 together with the carbons to which they are attached to form a six-membered lactone ring.
I
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The symbol R 3 represents a member selected from the group consisting of hydrogen,
Cualkyl, aryl, -OR 4 , -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen. Each
instance of the symbol R 4 independently represents a member selected from the group
consisting of hydrogen and Ci^alkyl.
5 [0087] In a preferred aspect of the present invention, the compound of Formula I has the
following structure:
In this case, the symbol R is a member selected from the group consisting of hydrogen,
Ci^alkoxy and Co-ealkoxy-aryl; the symbol R 2 is a member selected from the group
10 consisting of hydrogen and hydroxy; the symbol R 3 is a member selected from the group
consisting of hydrogen and -OR 4 ; and the symbol R 4 is Ci^alkyl.
[0088] In another preferred aspect of the present invention, the symbol R l is a member
selected from the group consisting of C^alkoxy, Co.6alkoxy-aryl and hydroxy. In a more
preferred aspect of the present invention, the symbol R 1 is a member selected from the group
15 consisting of hydroxy, methoxy and benzyloxy. In a most preferred aspect of the present
invention, the symbol R 1 is benzyloxy. In another preferred embodiment, the term aryl is a
member selected from the group consisting of phenyl and naphthyl.
[0089] In another aspect of the present invention, a pharmaceutical composition is
provided, comprising a pharmaceutical^ acceptable excipient and a compound, and all
20 pharmaceutical^ acceptable salts thereof, of Formula II:
In Formula II, the symbol R a is a member selected from the group consisting of hydrogen,
Ci^alkyl, aryl, -OR c , -NR*R C , -C0 2 R c , -C(0)R c , -QOJNR'R 6 , -CN, -N0 2 and halogen, while
the symbol R b is a member selected from the group consisting of:
R 3 n
25
WO 03/046207
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In the components above, the symbol X a represents a member selected from the group
consisting of O, S and NR C , while the symbol R c represents a member selected from the
group consisting of hydrogen, Ci. 6 alkyl and aryl optionally substituted with a member
selected from the group consisting of hydrogen, Ci- 6 atkyl> ^ -° Re > -NR C R C , -CN, -N0 2 and
5 halogen. The symbol R d represents a member selected from the group consisting of
hydrogen, Ct^alkyl, aryl, -OR e , -NR C R C and halogen. And, each instance of the symbol R c
independently represents a member selected from the group consisting of hydrogen and
Ci^alkyl.
[0090) In a preferred aspect of the present invention, Formula II has the following
10 structure:
ADMINISTRATION
[0091] An effective amount of the composition will be determined by the existence, nature,
15 and extent of any adverse side-effects that accompany the administration of the composition;
the LD50 of the composition; and the side-effects of the composition at various
concentrations. Typically, the amount of the composition administered will range from about
0.01 to about 20 mg per kg, more typically about 0.05 to about 1 5 mg per kg, even more
typically about 0.1 to about 10 mg per kg body weight.
20 [0092] The compositions can be administered, for example, by intravenous infusion, orally,
intraperitoneally, or subcutaneously. Oral administration is the preferred method of
administration. The formulations of compounds can be presented in unit-dose or multi-dose
sealed containers, such as ampoules and vials.
[0093] The compositions of the present invention are typically formulated with a
25 pharmaceutical^ acceptable carrier before administration to an individual or subject.
Pharmaceutically acceptable carriers are determined,, in part, by the particular composition
being administered, as well as by the particular method used to administer the composition.
Accordingly, there are a wide variety of suitable formulations of pharmaceutical
26
WO 03/046207 PCT/US02/38434
compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th
ed., 1989).
[0094] Formulations suitable for oral administration can consist of (a) liquid solutions, such
as an effective amount of the compound of Formula I or Formula II, suspended in diluents,
5 such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include
one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,
10 magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an
inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
15 containing, in addition to the active ingredient, carriers known in the art.
[0095] The compositions of the present invention may be in formulations suitable for other
routes of administration, such as, for example, intravenous infusion, intraperitoneally, or
subcutaneously. The formulations include, for example, aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes
20 that render the formulation isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. Injection solutions and suspensions can be prepared
from sterile powders, granules, and tablets.
[0096] The dose administered to a patient, in the context of the present invention should be
25 sufficient to effect a beneficial therapeutic response in the patient over time. For example, if
the compositions of the present invention are administered to treat or prevent cancer, such as
a tumor, the dose administered to the patient should be sufficient to prevent, retard, or reverse
tumor growth. The dose will be determined by the efficacy of the particular composition
employed and the condition of the patient, as well as the body weight or surface area of the
30 patient to be treated. The size of the dose also will be determined by the existence, nature,
and extent of any adverse side-effects that accompany the administration of a particular
composition in a particular patient.
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EXAMPLES
Compounds.
[0097] The compounds of the present invention can be synthesized by several methods
known to one of skill in the art. Methods for preparing the SI and S2 scaffolds are shown
5 below by way of example, and are by no means comprehensive of the methods that can be
used to synthesize the compounds of the present invention. One of skill in the art will
appreciate that the starting material, the reagants and the reactions shown in the schemes
below, can be appropriately modified in order to synthesize all the compounds of the present
invention. The appropriate modifications are known to those of skill in the art.
10
Yeast Media.
1 5 [0098] All strains can be grown in synthetic complete media (SC) or selective synthetic
drop-out media containing 2% glucose.
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Cell-based Chemical Screen for the Sir2p Inhibitors: Inhibition of Silencing Assays.
[0099) In order to find inhibitors of the deacetylase activity of Sir2p, screening was carried
out to identify compounds that perturbed silencing at each of the loci at which Sir2p is known
to act in S. cerevisiae: telomeres, HML, HMR, and the rDNA. The cell-based positive
5 selection screen was designed so that inhibition of Sir2p activity permitted normal cell
growth in order to avoid identifying cytotoxic compounds. Briefly, a yeast strain containing
a marker gene, such as a nutritional marker, in close proximity to a telomeres in S. cerevisiae
such that it is repressed by telomeric chromatin, is exposed to a test compound or set of test
compounds dissolved in DMSO and cultured under suitable conditions and in media
10 supplemented to permit growth only under conditions in which the marker gene is expressed.
After a suitable interval, the optical density of the culture is measured. An increase in optical
density corresponds to growth indicating a perturbation of silencing of the marker gene. All
strains in this and other examples herein can be grown in synthetic complete media (SC) or
selective synthetic drop-out media containing 2% glucose.
15 [0100] Initial screening for compounds that effect silencing was carried out using a URA3
assay. When the URA3 gene is in close proximity to a telomere in S. cerevisiae, it is
repressed by telomeric chromatin (Gottschling, D. E., et al (1990) Cell 63, 751-62).
Because Ura3p is required for uracil biosynthesis, cells with the silenced telomeric URA3
gene are unable to grow in media lacking uracil. Accordingly, genetic perturbation of
20 silencing activates URA3 expression and enables cells to grow in the absence of uracil
(Singer, M. S., et al. y (1998) Genetics 150, 613-3). Briefly, drug screening was performed in
96-well plates. Each well is inoculated with 150 jiL of yeast culture (strain: UCC2210 MATql
pprl adh4::URA3::TEL(VIhL)), containing 1 X 10 5 cell/ml in uracil-deficient media. A
library of 6000 compounds from the NCI repository was screened for those that disrupted
25 telomeric silencing. The compounds dissolved in DMSO are applied at three different
concentrations: 0.5, 5 and 50 ^M. Cultures are incubated for 36-48 h and growth in
individual wells is tested by optical density (OD 6 6o) measurements and visual inspection.
Eleven compounds identified in this primary screen were analyzed further to determine
whether silencing at the HML and HMR loci was also affected.
30 [0101] A secondary screening of the eleven identified compounds was carried out using a
TRP1 gene which utilized a yeast strain with a TRP1 gene integrated at the silent HMR locus
cannot grow in media lacking tryptophan (Buck, S. W. & Shore, D. (1995) Genes Dev. 9,
370-84). Using the method essentially described above, wells in a 96-well plate were
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inoculated with 150 nL of yeast culture (a yeast strain with a TRP1 gene integrated at the
silent HMR locus), containing 1 X 10 5 cell/ml in tryptophan-deficient media. The eleven
compounds dissolved in DMSO were added to the cells and the cultures are incubated for 36-
48 h and growth in individual wells is tested by optical density (OD 6 6o) measurements and
5 visual inspection. In this assay, one of the eleven compounds enabled cells to grow in media
lacking tryptophan (Fig. 15), indicating loss of silencing at HMR. This compound (1 ,2-
dihydro-3H-naphtho[2,l-b]pyran-3-one, Fig. 1/4), hereafter referred to as SI, also disrupted
silencing at HML.
Effect of SI on silencing at the HML a locus in MAT* cells.
1 0 [01 02] In one assay to confirm that S 1 was capable of inhibiting silencing in vivo, a
pheromone response assay was carried out. When haploid MAT* cells are exposed to the
mating pheromone a factor, they arrest in Gl phase of the cell cycle. Loss of silencing at the
.HMLa locus in MAT* cells results in expression of a mating type genes (Marsh, L., et al.
(1991) Annu. Rev. Cell Biol 7, 699-728). The coexpression of a and a genes creates a
15 pseudo-diploid state: cells are immune to a factor and unable to mate. In the presence of SI,
MAT* cells lost responsiveness to a factor (Fig. 1 Q and were defective for mating. Thus,
treatment with SI disrupted silencing at HML t HMR, and telomeric loci.
Effect of SI on Recombination at the rDNA locus.
[0103] Sir2p is involved in the silencing of rDNA through a protein complex known as
20 RENT (regulator of nuclear silencing and telophase exit), which does not include Sir3p or
Sir4p and acts at the ribosomal RNA gene cluster (rDNA). Silencing within the rDNA locus
is manifested in two ways. It can weakly repress expression of an inserted reporter gene
(Smith, J. S. & Boeke, J. D. (1997) Genes Dev. 11, 241-54), and it reduces recombination
between tandem copies of the ribosomal RNA genes (Gottlieb, S. & Esposito, R. E. (1989)
25 Cell 56, 771-6). Recombination was analyzed by measuring the loss rate of an ADE2 gene
integrated into the rDNA array essentially as described by Kaeberlein, M, et al (1999 Genes
Dev. 13, 2570-80). A logarithmic culture of a yeast strain containing an ADE2 gene
integrated into the rDNA array was exposed to 15 of SI or DMSO for six hours. After
six hours, the cultures were plated onto rich medium and the loss of expression of the ADE2
30 gene was measured and scored by the development of sectored red colonies. The results
showed that SI disrupted silencing of a reporter gene within the rDNA locus, just as it did at
telomeres and the HM loci. Treatment with SI increased recombination rate at the rDNA
locus seven-fold, which is similar to rates observed in a sir2 mutant (Fig. ID). There was no
30
WO 03/046207 PCT/US02/38434
effect on rDNA recombination in sir! cells treated with the compound, indicating that Si was
acting specifically through the SIR2 pathway.
Whole Gene Array Analysis For Transcriptional Profiling.
[01 04 J In addition to SIR2, the S. cerevisiae genome encodes four SIR2 homologues:
HST1-4 (Homologue of Sir Two) (Brachmann, C. B., et al (1995) Genes Dev. 9, 2888-902).
Hst2p is located in the cytoplasm and is responsible for virtually all the NAD + -dependent
deacetylase activity detected in a cellular lysate (Smith, J. S., et al, (2000) Proa Natl. Acad.
Sci. USA 97, 6658-63). Its relevant biological substrate is unknown. Hstlp is required for
transcriptional repression of meiotic genes (Xie, J., et al. (1999) EMBOJ. 18, 6448-54),
whereas little is known about the cellular function of Hst3p or Hst4p. In order to determine
whether the anti-silencing effects of SI were mediated solely by Sir2p, and. whether SI
affected any of the Hst proteins, the expression profile of wild type cells grown in the
presence of SI was compared to that of sir2, hstl, hst2, hst3 or hst4 deletion mutants by
whole genome DNA microarray analysis.
[0105] Strains for the DNA array experiments for the whole genome analysis were
obtained from Research Genetics (wild type BY4741 : MAT* his3, leu2, met 15, ura4 or
isogenic sir2, hstl, hst2, hst3 and hst4 deletion mutants). Several colonies from fresh
cultures were inoculated into synthetic complete medium with 2% glucose, grown overnight
at 30*C, diluted to 0.5-1 X 10 6 cell/ml and grown for an additional 6-9 hours until reaching a
density of 0.5-1 X 10 7 cells/ml. For experiments with SI, drug or the solvent (DMSO) was
added at the beginning of the final 9-hour growth phase. In experiments with cycloheximide,
cells were treated with 50 ng/ml of cycloheximide for 40 minutes prior to the addition of SI.
Total RNA was extracted using the hot acid phenol method. Microarray construction and
hybridization protocols were modified from those described elsewhere (DeRisi, J. L. et al.
(1997) Science 278, 680-6). Briefly, yeast microarrays were constructed employing a set of
-6200 orf-specific PCR primer pairs (Research Genetics, Huntsville, AL), which were used
to amplify each open reading frame (orf) of the yeast genome. Individual PCR products were
verified as unique via gel electrophoresis and purified using ARRAYIT 96-well PCR
purification kits (TeleChem International, Sunnyvale, CA). Purified PCR products were
mechanically "spotted" in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH
7.0) onto poly-lysine coated microscope slides using an OMNIGRID high-precision robotic
gridder (GeneMachines, San Carlo, CA).
10106] The protocol used for cDNA labeling is a modification of a protocol described
elsewhere (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm).
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Briefly, labeled cDNA targets were prepared by reverse transcription of 30 total RNA
using oligo dT(18) primer in the presence of 0.2 mM 5-(3-aminoallyl)-2Meoxyuridine-5'-
triphosphate (aa-dUTP; Sigma-Aldrich Company, St. Louis, MO), 0.3 mM dTTP, and 0.5
mM each of dATP, dCTP, and dGTP. Following cDNA synthesis, either Cy3 or Cy5 mono-
5 reactive fluors (Amersham Life Sciences, Arlington Heights, IL) were covalently coupled to
the cDNA-incorporated aminoallyl linker in the presence of 50 mM sodium bicarbonate (pH
9.0). Two color expression profiles were generated using microarrays in which reference and
experimental cDNA targets were labeled with different fluors. Following co-hybridization to
the chip, a fluorescent image of the microarray was collected at both emission wavelengths
10 using a GenePix 4000 fluorescent scanner (Axon Instruments, Inc., Foster City, CA) and
image analysis is performed using GenePix Pro Microarray Acquisition and Analysis
Software.
[0107] Three competitive hybridizations for each experimental group (sir2, hstl, hst2, hst3
and hst4 versus wild type, SI treated wild type versus wild type and SI plus cycloheximide
15 versus cycloheximide alone) were performed using three separate cultures and log2 of the
expression ratio calculated for every ORF. To assess the intrinsic variation of expression
level for different ORF's, nine wild type versus wild type hybridizations were performed
using nine separate cultures. Student t-test was used to assess if the difference between the
log 2 of the expression ratio for ORF in the experimental and control group (wild type versus
20 wild type) was significant.
[0108] Using this array method, the transcriptional effects of SI correlated most highly
with those of a sir2 mutation (correlation coefficient 0.748, Fig. 2A). Genes adjacent to
telomeres such as COS12, and the al and a2 genes from the HML locus, were significantly
up-regulated in both conditions (Fig. 2A). The expression of M/4 7a- specific (e.g. MFA1,
25 STE2, STE6, BAR1) and haploid-specific genes (e.g. FUSh STE5) was down-regulated in
both SI treated cells and in sir2 cells (Fig. 2, A and B). SI also up-regulated a small number
of genes that were not altered in sir2 cells, including meiosis specific genes (e.g. SPS1) which
appear to be regulated by HSTJ (Fig. 25). There was no overlap between SI and HST2,
HST3, or HST4 regulated genes (Fig. 2D). Thus the majority of all transcriptional changes
30 (88%) induced by SI were mediated through SIR2 and a smaller subset (9%) through HSTJ
(Fig. 25). These results indicate that SI is a selective Sir2p inhibitor.
[0109] Sir2p is critical for silencing, yet the majority of the transcriptional changes induced
by either chemical or genetic inactivation of the enzyme constituted transcriptional down-
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WO 03/046207 PCT/US02/38434
regulation (Fig. 2, A and B). A number of these changes are known to be indirect. For
instance, haploid specific genes are down-regulated by the gene products of the derepressed
HMLa and HMR* loci (Marsh, L., et al. (1991) Annu. Rev. Cell Biol. 7, 699-728). SI,
combined with the protein synthesis inhibitor cycloheximide, afforded an opportunity to
identify genes that are directly regulated by Sir2p. Such an examination has not been
possible before because conditional alleles of SIR2 are not available. The addition of
cycloheximide did not affect the upregulation of genes by SI treatment. In contrast, virtually
all transcriptional down-regulation was abolished in the absence of new protein synthesis.
This confirmed that the direct effect of Sir2p is to repress transcription (Fig. 2Q. With the
exception of a single gene, BPHJ 9 the only genes that were up-regulated as a result of Sir2p
inactivation in the presence of cycloheximide were subtelomeric genes and silent mating type
loci, indicating that Sir2p activity does not affect transcription outside of these regions.
Overall, these results are consistent with a recent study examining the location of Sir2p by
genome-wide chromatin immunoprecipitation (Lieb, J. D., et al (2001) Nat. Genet. 28, 327-
34).
HDA Assay for Determination of Inhibition of Deacetylase Activity of Sir2p by SI.
[01 10] Without being bound to any particular theory, the phenotypic changes caused by S 1
are thought to be the result of inhibition of the histone deacetylase activity of Sir2p.
Accordingly, SI was evaluated for its ability to inhibit the histone deacetylase activity of
Sir2p in vitro. An [ 3 H]-acetylated histone H4 peptide was used in the assay which measured
the NAD + -dependent release of free [ 3 H]-acetate in the presence of whole yeast cell extract
from an hst2 strain overexpressing yeast SIR2. A cell extract obtained from a SIR2
overexpressing hst2 strain had robust NAD + -dependent histone deacetylase activity derived
exclusively from Sir2p (Fig. 3, A and B).
[01 1 1 J Briefly, histone H4 was chemically acetylated using the HDAC Assay Kit (Upstate
Biotechnology). The whole cell extract is prepared as described (Smith, J. S. et al. (2000)
Proc. Natl. Acad. ScL USA 97, 6658-63) from an hst2A strain containing 2ji plasmid with
galactose-inducible wild type SIR2 (pAR14, Braunstein, M. et al. (1993) Genes Dev. 7, 592-
604) or mutant SIR2 (GAL-SW2-Y298N or GAL-S/tf 2-H286Q) or empty vector. For the
histone deacetylase assays, 50 \ig of yeast whole-cell protein extract was incubated with [ 3 H]
acetylated histone H4-peptide (40,000 cpm) with or without 500 nM NAD + in a 100 \il
reaction. The buffer contained 1 50 mM NaCl, 25 mM sodium phosphate pH 7.4 and 1 mM
DTT. Reactions were incubated at 30°C for 16 hours and were stopped by the addition of 25
33
WO 03/046207 PCT/US02/38434
ill of 1 N HC1 and 0.15 N acetic acid. Released [ 3 H] acetate was extracted with 400 \i\ of
ethyl acetate. SI induced dose dependent inhibition of histone deacetylase activity in the
yeast extract, with an IC50 of 60 fiM (Fig. 3A). This result established Sir2p deacetylase
activity as a direct target of SI .
5 Preparation and Identification of Mutations Conferring Drug Resistance to SI.
[01 12] To obtain further insight into the molecular mechanism by which SI inhibited
deacetylase activity of Sir2p, mutant forms of Sir2p were generated that were resistant to the
compound. The conserved core region of SIR2 was amplified using error prone PCR and was
integrated into a SIR2 containing centromeric plasmid (pRS3 \4-SIR2) by cotransformation
10 into a sir 2 A strain with a URA3 telomeric marker (strain AB14053 MATql sir 2 pprl
adh4::URA3::TEL(VTl-L)). Transformants from selective (-trp) media are pooled and
aliquots plated onto selective media containing 5-fluoroorotic acid (5-FOA) and 1 0 |iM S 1 .
Plasmid DNA was recovered from the individual colonies and was retransformed into the test
strain to assure that drug resistance was conferred by S7/?2-containing plasmid. The entire
15 SIR2 open reading frames from 20 independent plasmids conferring SI resistance were
sequenced. Mutations were introduced into a plasmid containing galactose inducible SIR2
(pAR14 (Braunstein, M. et ah (1993) Genes Dev. 7, 592-604)) using gap repair or site
directed mutagenesis to make GAL-SIR2-Y298N and GAL-SIR2-H286Q. Three alleles of
SIR2 (SIR2-H286Q, SIR2-L287Q and SIR2-Y298N)v/cre identified that render yeast cells
20 resistant to the anti-silencing effects of S 1. Silencing was at normal levels in the drug
resistant mutants in the absence of drug, but disruption of silencing in the mutants required
higher concentrations of SI than in wild type strains (Fig. 3C). In vitro, when compared to
equivalent amounts of wild type Sir2p, mutant proteins exhibited similar histone deacetylase
activity in the absence of drug, with increased resistance to the inhibitory effect of SI (Fig. £
25 AsndB).
[01 13] The three mutations lie in close proximity within a region that is highly similar to
human SIRT2. Most interestingly, the crystal structure of SIRT2 defines this region to be a
hydrophobic cavity that is hypothesized to be the binding site for acetylafed lysine peptides
(Finnin, M. S., et al (2001) Nat. Struct Biol. 8, 621-5 and Min, J., et ai (2001) Cell 105,
30 269-79.). As noted above, the expression profile of SI -treated cells had no overlap with
mutant hst2, hst3 f or hst4 strains, but did have some overlap with the hstl profile. Of all the
HST genes, Hstlp has the highest sequence similarity (86% identity) to Sir2p in the 50 amino
acid region containing the SI resistance mutations (Fig. 3D). Since Hstlp also acts to repress
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WO 03/046207 PCT/US02/38434
gene expression via hypoacetylation of histones (Rusche, L. N. & Rine, J. (2001) Genes Dev.
15, 955-67 and Sutton, A., et al (2001) Mol Cell Biol 21, 3514-22.), it seems likely that this
shared region defines a common binding pocket for acetylated histone tails in both proteins.
Thus, it appears that SI inhibits the deacetylase activity of Sir2p by blocking access to the
5 acetylated histone binding pocket, or by altering the conformation of the acetylated histone
binding pocket, such that the deacetylase activity of Sir2p is inhibited.
Continuous Deacetylase Activity of Sir2p is Required for the Maintenance of the Silent
State in Non-Dividing Cells.
[0114] The establishment of silencing in previously active chromatin is a cell cycle
10 dependent event that can be accomplished only during S-phase (Li, Y. C, et al (2001)
Science 291, 650-3 and Kirchmaier, A. L. & Rine, J. (2001) Science 291, 646-50). Once
established, the silent state needs to be maintained between cell divisions, after mitosis, in Gl
and into the next S phase. Studies with a temperature sensitive allele of SIR3 demonstrated
that silencing is quickly lost in Gl -arrested cells after cells are shifted to the nonpermissive
15 temperature (Miller, A. M. & Nasmyth, K. A. (1984) Nature 312, 247-51). In contrast,
removal of the DNA silencer elements from the HMLa locus in Gl -arrested cells does not
disrupt silencing (Holmes, S. G. & Broach, J. R. (1996) Genes Dev. 10, 1021-32). The study
with the temperature sensitive allele of SIR3 suggests that the presence of the entire SIR
complex is required for the maintenance of a silent state.
20 [01 1 5] The requirement for the deacetylase activity of Sir2p for the maintenance of a silent
state in non-dividing cells or whether Sir2p was dispensable once silent chromatin was
formed was not established. To address this issue, the ability of SI to inhibit the histone
deacetylase activity of Sir2p was used. Briefly, MAT* cells were first arrested in Gl using a
factor and then treated with SI . While untreated cells remained arrested in Gl, those treated
25 with S 1 progressed through the cell cycle (Fig. 4A). This was presumably due to loss of
mating competence, a consequence of expression of the a2 gene from the "silent" HML
locus. To test this idea more directly, a MAT* strain with a single Gl cyclin gene (CLN3) y
which is under control of a galactose-inducible promoter (Cross, F. R. (1990) Mol Cell Biol
10, 6482-90), was arrested in Gl by replacing galactose with glucose in the media. Once the
30 cells arrested in Gl, they were treated with SI or a DMSO control. While the cells remained
arrested in Gl under both conditions, a2 mRNA expression from HML was detected only in
the SI -treated cells (Fig. 45). The lag period of several hours between the application of SI
and the appearance of <x2 mRNA was similar to the delay before cell cycle progression was
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observed in the a factor arrested cells treated with SI (described above). These results
demonstrated that the deacetylase activity of Sir2p is continuously required for the
maintenance of the silent state in non-dividing cells.
[0116] As a result of these studies, Sir2p must remain diligent in maintaining the silent
5 state, in order to counteract the constant activity of histone acetylases. The acetylases may
gain access to the chromatin in a targeted manner via transcriptional activators (Aparicio, 0.
M. & Gottschling, D. E. (1994) Genes Dev. 8, 1133-46 and Sekinger, E. A. & Gross, D. S.
(2001) Cell 105, 403-14) or be part of a global histone acetylation maintenance system
(Vogelauer, M, et al. (2000) Nature 408, 495-8). These results also support the idea that
10 silent chromatin is not a static, rigid structure, but rather that it is in a dynamic equilibrium
with silencing factor exchanging on and off the chromatin, even when cells are not dividing
(Cheng, T. H. & Gartenberg, M R. (2000) Genes Dev. 14, 452-63).
[01 1 7] These results underscore the power of phenotypic screening in model systems to
identify new compounds that are useful for dissecting complex biological processes such as
15 silencing in vivo. To this end, the identification of an inhibitor of Sir2p complements the
existing inhibitors of histone deacetylases (i.e. trapoxin and trichostatin (Taunton, J., et al.
(1996) Science 272, 408-1 1)). In addition to histones, many other proteins are regulated by
acetylation, including pRb, E2F and p53 proteins (Chan, H.M., et al. (2001) Nat. Cell Biol.
3,667-74; Martinez-Balbas , M. A., et al. (2000) EMBO J. 19,662-7 1 ; and Luo, J., et al.
20 (2000) Nature 408, 377-81). Two recent reports (Vaziri, H., et al. (2001) Cell 107, 149-159
and Luo, J., et al (2001) Cell 107, 137-148) implicate deacetylation of p53, by Sir2, in
down-regulation of transcriptional and proapoptotic activities of p53 in response to DNA
damage. Toxicity assays using SI and a variety of DNA damaging agents have shown that
SI sensitizes mammalian cells to these agents, consistent with SI abrogating Sir2p activity on
25 p53. Thus, SI is a useful component in the evaluation of Sir2p-like deacetylases as drug
targets for treating cancer and other diseases (Wolffe, A. P. (2001) Oncogene 20, 2988-90
and Tycko, B. & Ashkenas, J. (2000) J. Clin. Invest 105, 245-6).
SI Sensitization of Mammalian Cells to DNA Damage.
[0118] To test whether SI could sensitize cells to DNA damage, mammalian cells were
30 exposed to etoposide, a DNA damaging agent, in the presence and absence of SI. Briefly,
Rati a and primary human fibroblasts (HFF) were treated with etoposide alone (0) or with SI
(25, 50 and 100 nM) and etoposide for 72 hours. Viability of cells was assessed with 3H-
thymidine incorporation. Viability of cells in etoposide relative to viability without etoposide
36
WO 03/046207 PCT/US02/38434
is graphed for every concentration of S 1 , In both Rat 1 a cells and human foreskin fibroblast
cells, Si induced a dose-dependent sensitization to etoposide (Fig. 5).
[01 19] Figure 6 is a table of compounds useful in the present invention, and their activity as
determined in the assays described above.
5
[0120] Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be obvious that
certain changes and modifications may be practiced within the scope of the appended claims.
In addition, each reference provided herein is incorporated by reference in its entirety to the
10 same extent as if each reference was individually incorporated by reference.
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WHAT IS CLAIMED IS:
1 1 . A method for identifying compounds useful for the treatment of cancer
2 or genetic blood diseases, comprising the step of determining whether the compound inhibits
3 the deacetylase activity of a NAD + -dependent deacetylase.
1 2. The method of claim 1, wherein said method is for identifying
2 compounds which will be useful for the treatment of silenced tumor suppressor genes, B-cell-
3 derived non-Hodgkin lymphomas and diffuse large B-cell lymphomas.
1 3 . The method of claim 1 , wherein said method is for identifying
2 compounds which will be useful for the treatment of thalassaemias and sickle cell disease.
1 4. The method of claim 1 , wherein said NAD + -dependent deacetylase is a
2 member of the SIR2 family of proteins.
1 5. The method of claim 4, wherein said step of determining comprises the
2 step of specifically binding radiolabeled (1 ,2-dihydro-3H-naphtho[2, l-b]pyran-3-one) to the
3 ligand binding domain of a member of the SIR2 family of proteins.
1 6. The method of claim 4, wherein said member of the SIR2 family of
2 proteins is selected from the group consisting of Sir2p and SIR2o£.
1 7. The method of claim 6, wherein said member of the SIR2 family of
2 proteins is SIR2a.
1 8. A method for treating cancer or genetic blood diseases, comprising the
2 step of administering to a subject in need thereof, a therapeutically effective amount of a
3 compound identified by the method of claim 1.
1 9. The method of claim 8, wherein said method is for the treatment of
2 silenced tumor suppressor genes, B-cell-derived non-Hodgkin lymphomas and diffuse large
3 B-cell lymphomas.
1 10. The method of claim 8, wherein said method is for treating
2 thalassaemias and sickle cell disease.
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1 11. The method of claim 8, wherein said compound is an inhibitor of the
2 NAD + -dependent deacetyiase activity of a member of the SIR2 family of proteins.
1 12. The method of claim 1 1 , wherein said member of the SIR2 family of
2 proteins is selected from the group consisting of Sir2p and SIR2a
1 13. The method of claim 1 1 , wherein said member of the SIR2 family of
2 proteins is SIR2a.
1 14. A method for identifying compounds which will be useful for the
2 treatment of cancer or genetic blood diseases, comprising the step of determining whether the
3 compound inhibits the NAD + -dependent deacetyiase activity of a member of the SIR2 family
4 of proteins.
1 15. The method of claim 14, wherein said method is for identifying
2 compounds which will be useful for the treatment of silenced tumor suppressor genes, B-cell-
3 derived non-Hodgkin lymphomas and diffuse large B-cell lymphomas.
1 1 6. The method of claim 14, wherein said method is for identifying
2 compounds which will be useful for the treatment of thalassaemias and sickle cell disease.
1 1 7. The method of claim 14, wherein said step of determining comprises
2 the step of specifically binding radiolabeled (l,2-dihydro-3H-naphtho[2,l-b]pyran-3-one) to
3 the ligand binding domain of a member of the SIR2 family of proteins.
1 1 8. The method of claim 17, wherein said member of the SIR2 family of
2 proteins is selected from the group consisting of Sir2p and SIR2a
1 19. The method of claim 17, wherein said member of the SIR2 family of
2 proteins is SIR2a.
1 20. A method for treating cancer or genetic blood diseases, comprising the
2 step of administering to a subject in thereof, a therapeutically effective amount of a
3 compound which was identified by the method of claim 14.
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1 21. The method of claim 20, wherein said method is for the treatment of
2 silenced tumor suppressor genes, B-cell-derived non-Hodgkin lymphomas and diffuse large
3 B-cell lymphomas.
1 22. The method of claim 20, wherein said method is for treating
2 thalassaemias and sickle cell disease.
1 23. The method of claim 20, wherein said compound is an inhibitor of the
2 NAD + -dependent deacetylase activity of a member of the SIR2 family of proteins.
1 24. The method of claim 23, wherein said member of the SIR2 family of
2 proteins is selected from the group consisting of Sir2p and SIR2qj.
1 25. The method of claim 23, wherein said member of the SIR2 family of
2 proteins is SIR2a
1 26. A method for activating a silenced gene in a cell, comprising
2 contacting said cell with an effective amount of a compound capable of inhibiting the
3 NAD + -dependent deacetylase activity of a member of the SIR2 family of proteins.
1 27. A method for promoting the p53-dependent apoptosis of a cell,
2 comprising contacting said cell with an effective amount of a compound capable of inhibiting
3 the NAD + -dependent deacetylase activity of a member of the SIR2 family of proteins.
1 28. A method for inhibiting BCL6 transcriptional repressor activity,
2 comprising contacting a cell with an effective amount of a compound capable of inhibiting
3 the NAD + -dependent deacetylase activity of a member of the SIR2 family of proteins.
1 29. A method for inhibiting the deacetylase activity of a NAD + -dependent
2 deacetylase, said method comprising contacting said NAD + -dependent deacetylase with a
3 NAD + -dependent deacetylase inhibiting amount of a compound of Formula I:
X
4 R 2
5 wherein
40
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6 X is a member selected from the group consisting of O and S;
7 L 1 and L 2 are each members independently selected from the group consisting of O, S,
8 ethylene and propylene, substituted with 0-2 R groups wherein each R group
9 is independently a member selected from the group consisting of C\ ^alkyl,
10 C 2 -6aIkenyl and -C0 2 R 4 , and wherein exactly one of L 1 and L 2 is selected from
1 1 the group consisting of O and S;
12 R'andR 2 are each members independently selected from the group consisting of
13 hydrogen, Ci^alkoxy, Co-6alkoxy-aryl and hydroxy; or
14 R and R are taken together with the carbons to which they are attached to form a
1 5 six-membered lactone ring;
16 R 3 is a member selected from the group consisting of hydrogen, Ci- 6 alkyl, aryl, -OR 4 ,
17 -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen; and
1 8 each instance of R 4 is independently a member selected from the group consisting of
1 9 hydrogen and C i ^alkyl.
1 30. The method of claim 29, wherein said compound has the formula:
R
2 R*
3 wherein
4 R is a member selected from the group consisting of hydrogen, Ci. 6 alkoxy and
5 Co^alkoxy-aryl;
6 R 2 is a member selected from the group consisting of hydrogen and hydroxy;
7 R 3 is a member selected from the group consisting of hydrogen and -OR 4 ; and
8 R 4 isC,^alkyL
1 31. The method of claim 29, wherein
2 R 1 is a member selected from the group consisting of C^alkoxy, Co-6alkoxy-aryl and
3 hydroxy.
1 32. The method of claim 3 1 , wherein said aryl is a member selected from
2 the group consisting of phenyl and naphthyl.
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1 33. The method of claim 29, wherein
2 R 1 is a member selected from the group consisting of hydroxy, methoxy and
3 benzyloxy.
1 34. The method of claim 29, wherein
2 R 1 is benzyloxy.
1 35. A method for inhibiting the deacetylase activity of a NAD + -dependent
2 deacetylase, said method comprising contacting said NAD + -dependent deacetylase with a
3 NAD + -dependent deacetylase inhibiting amount of a compound of Formula II:
R b
R T II n
5 wherein
6 R a is a member selected from the group consisting of hydrogen, d^alkyl, aryl, -OR e ,
7 -NRTl 6 , -C0 2 R e , -C(0)R e , -CCOJNR'R 6 , -CN, -N0 2 and halogen;
8 R b is a member selected from the group consisting of:
9 O ;
10 X a is a member selected from the group consisting of O, S and NR e ;
11 R c is a member selected from the group consisting of hydrogen, Ci- 6 alkyl and aryl
12 optionally substituted with a member selected from the group consisting of
13 hydrogen, Ci- 6 alkyl, aryl, -OR c , -NR e R e , -CN, -N0 2 and halogen;
14 R d is a member selected from the group consisting of hydrogen, Ci_ 6 alkyl, aryl, -OR e ,
1 5 -NR e R c and halogen; and
16 each instance of R c is independently a member selected from the group consisting of
1 7 hydrogen and Ci^alkyl.
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1 36, The method of claim 35, wherein said compound has the formula:
2
1 37. A method for the treatment of cancer, comprising the step of
2 administering to a subject in need of such treatment:
3 i) a first amount of an antineoplastic agent; and
4 ii) a second amount of a compound of Formula I:
X
R 3
5
6 wherein
7 X is a member selected from the group consisting of O and S;
8 L 1 and L 2 are each members independently selected from the group consisting of O, S,
9 ethylene and propylene, substituted with 0-2 R groups wherein each R group
1 0 is independently a member selected from the group consisting of Chalky!,
1 1 C 2 . 6 alkenyl and -C0 2 R 4 , and wherein exactly one of L l and L 2 is selected from
1 2 the group consisting of O and S;
13 R 1 and R 2 are each members independently selected from the group consisting of
14 hydrogen, C^alkoxy, Co-6alkoxy-aryl and hydroxy; or
15 R l and R 2 are taken together with the carbons to which they are attached to form a
1 6 six-membered lactone ring;
17 R 3 is a member selected from the group consisting of hydrogen, Chalky!, aryl, -OR 4 ,
1 8 -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen; and
19 each instance of R 4 is independently a member selected from the group consisting of
20 hydrogen and C i ^alkyl .
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1
38. The method of claim 37, wherein said antineoplastic agent is a member
2 selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites,
3 antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological
4 response modifiers, DNA-alkylating agents, DNA intercaiators, DNA repair agents, growth
5 factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disruptors
6 and topoisomerase I/II inhibitors.
1 39. The method of claim 37, wherein said antineoplastic agent is a member
2 selected from the group consisting of cyclophosphamide, triethylenephosphoramide,
3 triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine,
- 4 trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU,
5 benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil,
6 cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin,
7 estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide,
8 improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,
9 novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen,
10 temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
1 1 vincristine, vinorelbine and vindesine.
1 40. The method of claim 37, wherein said antineoplastic agent is
2 administered after said compound.
1 41 . The method of claim 37, wherein said antineoplastic agent is
2 administered simultaneously with said compound.
1 42. The method of claim 37, wherein said antineoplastic agent is
2 administered prior to said compound.
1 43. A method for the treatment of cancer, comprising the step of
2 administering to a subject in need of such treatment:
3 i) a first amount of an antineoplastic agent; and
4 • ii) a second amount of a compound of Formula II:
5
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6 wherein
7 R a is a member selected from the group consisting of hydrogen, C|. 6 alkyl, aryl, -OR e ,
8 -NR e R e , -C0 2 R e , -C(0)R e , -C(0)NR e R e , -CN, -N0 2 and halogen;
9 R" is a member selected from the group consisting of:
10 ^ I ;
11 X a is a member selected from the group consisting of O, S and NR C ;
12 R c is a member selected from the group consisting of hydrogen, Ci- 6 alkyl and aryl
13 optionally substituted with a member selected from the group consisting of
14 hydrogen, Ci^alkyl, aryl, -OR e , -NR e R c , -CN, -N0 2 and halogen;
15 R d is a member selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR e ,
1 6 -NR e R c and halogen; and
1 7 each instance of R e is independently a member selected from the group consisting of
1 8 hydrogen and C i ^alkyl .
1 44. The method of claim 43, wherein said antineoplastic agent is a member
2 selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites,
3 antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological
4 response modifiers, DNA-alkylating agents, DNA intercalators, DNA repair agents, growth
5 factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disruptors
6 and topoisomerase I/II inhibitors.
1 45. The method of claim 43, wherein said antineoplastic agent is a member
2 selected from the group consisting of cyclophosphamide, triethylenephosphoramide,
3 triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine,
4 trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU,
5 benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil,
6 cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin,
7 estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide,
8 improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,
9 novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen,
10 temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
1 1 vincristine, vinorelbine and vindesine.
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1 46. The method of claim 43, wherein said antineoplastic agent is
2 administered after said compound.
1 47. The method of claim 43, wherein said antineoplastic agent is
2 administered simultaneously with said compound.
1 48. The method of claim 43, wherein said antineoplastic agent is
2 administered prior to said compound.
1 49. A composition for the treatment of cancer, comprising an
2 antineoplastic agent and a compound of Formula I:
• X
R3fn i
3 R 2
4 wherein
5 X is a member selected from the group consisting of O and S;
6 L 1 and L 2 are each members independently selected from the group consisting of O, S,
7 ethylene and propylene, substituted with 0-2 R groups wherein each R group
8 is independently a member selected from the group consisting of Ci^alkyl,
9 C2-6alkenyl and -CO2R 4 , and wherein exactly one of L 1 and L 2 is selected from
10 the group consisting of O and S;
11 R 1 and R 2 are each members independently selected from the group consisting of
12 hydrogen, Ci^alkoxy, C 0 -6alkoxy-aryl and hydroxy; or
13 R 1 and R 2 are taken together with the carbons to which they are attached to form a
14 six-membered lactone ring;
15 R 3 is a member selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR 4 ,
16 -NR 4 R 4 , -C0 2 R\ -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen; and
17 each instance of R 4 is independently a member selected from the group consisting of
18 hydrogen and Ci^alkyl.
1 50. The composition of claim 49, wherein said antineoplastic agent is a
2 member selected from the group consisting of antiangiogenic and antivascular agents,
3 antimetabolites, antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides,
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4 biological response modifiers, DNA-alkylating agents, DNA intercalators, DNA repair
5 agents, growth factor receptor kinase inhibitors, hormone agents, immunoconjugates,
6 microtubule disruptors and topoisomerase I/II inhibitors.
2 member selected from the group consisting of cyclophosphamide, triethylenephosphoramide,
3 triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine,
4 trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU,
5 benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil,
6 cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin,
7 estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide,
8 improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,
9 novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen,
10 temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
1 1 vincristine, vinorelbine and vindesine.
1 52. The composition of claim 49, wherein said antineoplastic agent is
2 administered after said compound.
1 53. The composition of claim 49, wherein said antineoplastic agent is
2 administered simultaneously with said compound.
1 54. The composition of claim 49, wherein said antineoplastic agent is
2 administered prior to said compound.
1 55. A composition for the treatment of cancer, comprising an
51. The composition of claim 49, wherein said antineoplastic agent is a
2 antineoplastic agent and a compound of Formula II:
3
4
II
wherein
6
5
R a is a member selected from the group consisting of hydrogen, Ci. 6 alkyl, aryl, -OR e ,
-NR C R C , -C0 2 R c , -C(0)R e , -C(0)NR e R € , -CN, -N0 2 and halogen;
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7 R b is a member selected from the group consisting of:
8 O ;
9 X a is a member selected from the group consisting of O, S and NR e ;
10 R c is a member selected from the group consisting of hydrogen, Ci_ 6 alkyl and aryl
1 1 optionally substituted with a member selected from the group consisting of
1 2 hydrogen, Ct^alkyl, aryl, -OR c , -NR e R e , -CN, -N0 2 and halogen;
13 R d is a member selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR c ,
14 -NR e R e and halogen; and
1 5 each instance of R e is independently a member selected from the group consisting of
1 6 hydrogen and Ci ^alkyl.
1 56. The composition of claim 55, wherein said antineoplastic agent is a
2 member selected from the group consisting of antiangiogenic and antivascular agents,
3 antimetabolites, antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides,
4 biological response modifiers, DNA-alkylating agents, DNA intercalators, DNA repair
5 agents, growth factor receptor kinase inhibitors, hormone agents, immunoconjugates,
6 microtubule disruptors and topoisomerase I/II inhibitors.
1 57. The composition of claim 55, wherein said antineoplastic agent is a
2 member selected from the group consisting of cyclophosphamide, triethylenephosphoramide,
3 triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine,
4 trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU,
5 benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil,
6 cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin,
7 estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide,
8 improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,
9 novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen,
10 temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine,
1 1 vincristine, vinorelbine and vindesine.
1 58. The composition of claim 55, wherein said antineoplastic agent is
2 administered after said compound.
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1 59. The composition of claim 55, wherein said antineoplastic agent is
2 administered simultaneously with said compound.
1 60. The composition of claim 55, wherein said antineoplastic agent is
2 administered prior to said compound.
1 61 . A pharmaceutical composition comprising a pharmaceutically
2 acceptable excipient and a compound of Formula I:
X
R 3
3
4 wherein
5 X is a member selected from the group consisting of 0 and S;
6 L 1 and L 2 are each members independently selected from the group consisting of 0, S,
7 ethylene and propylene, substituted with 0-2 R groups wherein each R group
8 is independently a member selected from the group consisting of Ci^alkyl,
9 C2-$alkenyl and -CO2R 4 , and wherein exactly one of L l and L 2 is selected from
10 the group consisting of O and S;
11 R*andR 2 are each members independently selected from the group consisting of
12 hydrogen, C^alkoxy, Co-6alkoxy-aryl and hydroxy; or
13 R l and R 2 are taken together with the carbons to which they are attached to form a
14 six-membered lactone ring;
15 R 3 is a member selected from the group consisting of hydrogen, d^alkyl, aryl, -OR 4 ,
1 6 -NR 4 R 4 , -C0 2 R 4 , -C(0)R 4 , -C(0)NR 4 R 4 , -CN, -N0 2 and halogen;
1 7 each instance of R 4 is independently a member selected from the group consisting of
1 8 hydrogen and C 1 -6alkyl ;
19 and all pharmaceutically acceptable salts thereof.
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1 62. The pharmaceutical composition of claim 61, wherein said compound
2 has the formula:
R
3 R 2
4 wherein
5 R 1 is a member selected from the group consisting of hydrogen, Cj^alkoxy and
6 Co-6alkoxy-aryl;
7 R 2 is a member selected from the group consisting of hydrogen and hydroxy;
8 R 3 is a member selected from the group consisting of hydrogen and -OR 4 ; and
9 R 4 is C^alkyl.
1 63. The pharmaceutical composition of claim 61 , wherein
2 R 1 is a member selected from the group consisting of Ci- 6 alkoxy, C 0 ^alkoxy-aryl and
3 hydroxy.
1 64. The pharmaceutical composition of claim 63, wherein said aryl is a
2 member selected from the group consisting of phenyl and naphthyl.
1 65 . The pharmaceutical composition of claim 6 1 , wherein
2 R l is a member selected from the group consisting of hydroxy, methoxy and
3 benzyloxy.
1 66. The pharmaceutical composition of claim 6 1 , wherein
2 R 1 is benzyloxy.
1 67. A pharmaceutical composition comprising a pharmaceutically
2 acceptable excipient and a compound of Formula II:
3
4 wherein
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WO 03/046207 PCT/US02/38434
5 R a is a member selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR c ,
6 -NR C R C , -C0 2 R e , -C(0)R e , -C(0)NR e R c , -CN, -N0 2 and halogen;
7 R b is a member selected from the group consisting of:
H
8 O ;
9 X a is a member selected from the group consisting of O, S and NR C ; .
10 R c is a member selected from the group consisting of hydrogen, Ci^alkyl and aryl
1 1 optionally substituted with a member selected from the group consisting of
12 hydrogen, Ci^alkyl, aryl, -OR e , -NR e R e , -CN, -N0 2 and halogen;
13 R d is a member selected from the group consisting of hydrogen, Ci^alkyl, aryl, -OR e ,
14 -NR*R C and halogen;
15 each instance of R c is independently a member selected from the group consisting of
1 6 hydrogen and Ci -6alkyl;
1 7 and all pharmaceutical^ acceptable salts thereof.
1 68. The pharmaceutical composition of claim 67, wherein said compound
2 has the formula:
3
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Figure 1 .
C D
1/6
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Figure 2.
2/6
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3/6
WO 03/046207
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Figure 4.
A
B
MAT*+S <p <y OMSO
0 1 4 8 >^ ^ 1 4 6
4/6
WO 03/046207
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cpm (%)
HFF
31 pM
Etoposide
lb
100
Vobo
Etoposide
r Toooo
Figure 5.
5/6
WO 03/046207
Figure 6.
PCT/US02/38434
Compound
Structure
Activity"
Compound
Structure
Activity 8
SI
+
Slh
MeO
+
Sla
+
Sli
4-+
Sib
++
Slj
++
Sic
Et0 2 C. )
fTT°
++
Slk
++
Sid
++
Sll
OMe
+
Sle
o
Q A^C0 2 Et
+
Sim
u
+++
Slf
0
1
++
Sin
+++
Slg
+++
'"+": IC 50 greater than 50 jiM; "++" : IC 50 less than 50 uM but greater than 20 uM; "+++": I C $0 less than
20 mM.
6/6
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